The present invention relates to highly selective phosphodiesterase (PDE) enzyme inhibitors and to their use in pharmaceutical articles of manufacture. In particular, the present invention relates to potent inhibitors of cyclic guanosine 3xe2x80x2,5xe2x80x2-monophosphate specific phosphodiesterase type 5 (PDE5) that when incorporated into a pharmaceutical product are useful for the treatment of sexual dysfunction. The articles of manufacture described herein are characterized by selective PDE5 inhibition, and accordingly, provide a benefit in therapeutic areas where inhibition of PDE5 is desired, with minimization or elimination of adverse side effects resulting from inhibition of other phosphodiesterase enzymes.
The biochemical, physiological, and clinical effects of cyclic guanosine 3xe2x80x2,5xe2x80x2-monophosphate specific phosphodiesterase (cGMP-specific PDE) inhibitors suggest their utility in a variety of disease states in which modulation of smooth muscle, renal, hemostatic, inflammatory, and/or endocrine function is desired. Type 5 cGMP-specific phosphodiesterase (PDE5) is the major cGMP hydrolyzing enzyme in vascular smooth muscle, and its expression in penile corpus cavernosum has been reported (Taher et al., J. Urol., 149:285A (1993)). Thus, PDE5 is an attractive target in the treatment of sexual dysfunction (Murray, DNandP 6(3):150-56 (1993)).
A pharmaceutical product, which provides a PDE5 inhibitor, is currently available and marketed under the trademark VIAGRA(copyright). The active ingredient in VIAGRA(copyright) is sildenafil. The product is sold as an article of manufacture including 25, 50, and 100 mg tablets of sildenafil and a package insert. The package insert provides that sildenafil is a more potent inhibitor of PDE5 than other known phosphodiesterases (greater than 80 fold for PDE1 inhibition, greater than 1,000 fold for PDE2, PDE3, and PDE4 inhibition). The IC50 for sildenafil against PDE5 has been reported as 3 nM (Drugs of the Future, 22(2), pp. 128-143 (1997)), and as 3.9 nM (Boolell et al., Int. J. of Impotence Res., 8 p. 47-52 (1996)). N.C. Sildenafil is described as having a 4,000-fold selectivity for PDE5 versus PDE3, and only a 10-fold selectivity for PDE5 versus PDE6. Its relative lack of selectivity for PDE6 is theorized to be the basis for abnormalities related to color vision.
While sildenafil has obtained significant commercial success, it has fallen short due to its significant adverse side effects, including facial flushing (10% incidence rate). Adverse side effects limit the use of sildenafil in patients suffering from visual abnormalities, hypertension, and, most significantly, by individuals who use organic nitrates (Welds et al., Amer. J. of Cardiology, 83 (5A), pp. 21(C)-28(C) (1999)).
The use of sildenafil in patients taking organic nitrates is believed to cause a clinically significant drop in blood pressure which could place the patient in danger. Accordingly, the package label for sildenafil provides strict contraindications against its use in combination with organic nitrates (e.g., nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, erythrityl tetranitrate) and other nitric oxide donors in any form, either regularly or intermittently, because sildenafil potentiates the hypotensive effects of nitrates. See C. R. Conti et al., Amer. J. of Cardiology, 83(5A), pp. 29C-34C (1999). Thus, even with the availability of sildenafil, there remains a need to identify improved pharmaceutical products that are useful in treating sexual dysfunction.
The present invention provides an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and an oral dosage form comprising a selective PDE5 inhibitor at unit dosages between about 1 and about 20 mg/dosage form. The beneficial effects of the present invention were observed in clinical studies and through the discovery that a selective PDE5 inhibitor meeting the following criteria allows for the effective oral administration of about 1 to about 20 mg/dosage form without contraindications generally required for PDE5 inhibitor products, such as warnings directed to vision abnormalities. A selective PDE5 inhibitor of the present invention exhibits:
1) at least a 100 fold differential in the IC50 values for the inhibition of PDE5 versus PDE6 for a particular PDE5 inhibitor (i.e., the IC50 value versus PDE5 is at least 100 times less than the IC50 value versus PDE6);
2) at least a 1000 fold differential in the IC50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC50 value less than 10 nM.
Significantly, clinical studies also revealed that an effective product having a reduced tendency to cause flushing in susceptible individuals can be provided. Most unexpectedly, the product also can be administered with clinically insignificant side effects associated with the combined effects of a PDE5 inhibitor and an organic nitrate. Thus, the contraindication once believed necessary for a product containing a PDE5 inhibitor is unnecessary when a selective PDE5 inhibitor, as defined above, is used as disclosed herein. Thus, the present invention provides an effective therapy for sexual dysfunction in individuals who previously were untreatable or suffered from unacceptable side effects, including individuals having cardiovascular disease, such as in individuals requiring nitrate therapy, having suffered a myocardial infarction more than three months before the onset of sexual dysfunction therapy, and suffering from class 1 congestive heart failure as defined by the New York Heart Association (NYHA), or individuals suffering from vision abnormalities.
The present invention provides an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor per dosage form.
The present invention further provides a method of treating conditions where inhibition of PDE5 is desired, which comprises administering to a patient in need thereof an oral dosage form containing about 1 to about 20 mg of a selective PDE5 inhibitor, as needed, up to a total dose of 20 mg/-day. The invention further provides the use of an oral dosage form comprising a selective PDE5 inhibitor at a dosage of about 1 to about 20 mg for the treatment of sexual dysfunction.
Specific conditions that can be treated by the method and article of the present invention, include, but are not limited to, male erectile dysfunction and female sexual dysfunction, particularly female arousal disorder, also known as female sexual arousal disorder.
In particular, the present invention provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC50 values for the inhibition of PDE5 versus PDE6,
(ii) at least a 1000 fold differential in IC50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC50 less than 10 nM, and
(iv) sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof, and that is free of contradictions associated with administration of organic nitrates; and
(c) a container.
The present invention further provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC50 values for the inhibition of PDE5 versus PDE6,
(ii) at least a 1000 fold differential in IC50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC50 less than 10 nM, and
(iv) a sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof and that is using an organic nitrate; and
(c) a container.
The present invention also provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC50 values for the inhibition of PDE5 versus PDE6,
(ii) at least 1000 fold differential in IC50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC50 less than 10 nM, and
(iv) a sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof and that is suffering from a condition selected from the group consisting of a retinal disease, proneness to flushing, proneness to vision abnormalities, class 1 congestive heart failure, a myocardial infarction 90 days or more before onset of the sexual dysfunction treatment, and combinations thereof; and
(c) a container.
For purposes of the present invention as disclosed and described herein, the following terms and abbreviations are defined as follows.
The term xe2x80x9ccontainerxe2x80x9d means any receptacle and closure therefor suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.
The term xe2x80x9cIC50xe2x80x9d is the measure of potency of a compound to inhibit a particular PDE enzyme (e.g., PDE1c, PDE5, or PDE6). The IC50 is the concentration of a compound that results in 50% enzyme inhibition in a single dose-response experiment. Determining the IC50 value for a compound is readily carried out by a known in vitro methodology generally described in Y. Cheng et al., Biochem. Pharmacol., 22, pp. 3099-3108 (1973).
The term xe2x80x9cpackage insertxe2x80x9d means information accompanying the product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the xe2x80x9clabelxe2x80x9d for a pharmaceutical product.
The term xe2x80x9coral dosage formxe2x80x9d is used in a general sense to reference pharmaceutical products administered orally. Oral dosage forms are recognized by those skilled in the art to include such forms as liquid formulations, tablets, capsules, and gelcaps.
The term xe2x80x9cselective PDE5 inhibitorxe2x80x9d is defined as a PDE5 inhibitor having:
1) an IC50 value for the inhibition of PDE5 at least 100 times less than the IC50 value for the inhibition of PDE6;
2) an IC50 value for the inhibition of PDE5 at least 1,000 times less than the IC50 value for the inhibition of PDE1c; and
3) an IC50 value for the inhibition of PDE5 less than 10 nM. Selective PDE5 inhibitors vary significantly in chemical structure, and their use in the present invention is not dependent on chemical structure, but rather on the selectivity and potency parameters disclosed herein.
The term xe2x80x9cvision abnormalitiesxe2x80x9d means abnormal vision characterized by blue-green vision believed to be caused by PDE6 inhibition.
The term xe2x80x9cflushingxe2x80x9d means an episodic redness of the face and neck attributed to vasodilation caused by the ingestion of a drug, usually accompanied by a feeling of warmth over the face and neck and sometimes accompanied by perspiration.
The term xe2x80x9cfree drugxe2x80x9d means solid particles of drug not intimately embedded in a polymeric co-precipitate.
As previously stated, the present invention is directed to an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and a dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor per unit dosage form. A selective PDE5 inhibitor useful in the present invention is a PDE5 inhibitor having:
1) at least a 100 fold differential in IC50 values for the inhibition of PDE5 versus PDE6;
2) at least a 1000 fold differential in IC50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC50 value less than 10 nM; and is sufficiently bioavailable to be effective in about 1 to about 20 mg unit dosages.
The differential is expressed as a PDE6/PDE5 ratio of IC50 values, i.e., the ratio of the IC50 value versus PDE6 to the IC50 value versus PDE5 (PDE6/PDE5) is greater than 100, more preferably greater than 300, and most preferably greater than 500.
Similarly, the ratio of IC50 value versus PDE1c to IC50 value versus PDE5 (PDE1c/PDE5) is greater than 1000. Preferred PDE5 inhibitors have a greater than 3,000 fold differential between the inhibition of PDE5 and PDE1c, more preferably greater than a 5,000 fold differential between IC50 value versus PDE5 and PDE1c. The potency of the inhibitor, as represented by the IC50 value versus PDE5, is less than 10 nM, preferably less than 5 nM, more preferably less than 2 nM, and most preferably less than 1 nM.
The package insert provides a description of how to administer a pharmaceutical product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding the use of the product. The package insert generally is regarded as the label of the pharmaceutical product. The package insert incorporated into the present article of manufacture indicates that the selective PDE5 inhibitor is useful in the treatment of conditions wherein inhibition of PDE5 is desired. The package insert also provides instructions to administer one or more about 1 to about 20 mg unit dosage forms as needed, up to a maximum total dose of 20 mg per day. Preferably, the dose administered is about 5 to about 20 mg/day, more preferably about 5 to about 15 mg, and most preferably an about 5 mg or about 10 mg dosage form administered once per day, as needed.
Preferred conditions to be treated include sexual dysfunction (including male erectile dysfunction; and female sexual dysfunction, and more preferably female arousal disorder (FAD)). The preferred condition to be treated is male erectile dysfunction.
Significantly, the package insert supports use of the product to treat sexual dysfunction in patients suffering from a retinal disease, for example diabetic retinopathy or retinitis pigmentosa, or in patients who are using organic nitrates. Thus, the package insert preferably is free of contraindications associated with these conditions, and particularly the administration of the dosage form with an organic nitrate. More preferably, the package insert also is free of any cautions or warnings both associated with retinal diseases, particularly retinitis pigmentosa, and associated with individuals prone to vision abnormalities. Preferably, the package insert also reports incidences of flushing below 2%, preferably below 1%, and most preferably below 0.5%, of the patients administered the dosage form. The incidence rate of flushing demonstrates marked improvement over prior pharmaceutical products containing a PDE5 inhibitor.
The container used in the present article of manufacture is conventional in the pharmaceutical arts. Generally, the container is a blister pack, foil packet, glass or plastic bottle and accompanying cap or closure, or other such article suitable for use by the patient or pharmacist. Preferably, the container is sized to accommodate 1-1000 solid dosage forms, preferably 1 to 500 solid dosage forms, and most preferably, 5 to 30 solid dosage forms.
Oral dosage forms are recognized by those skilled in the art to include, for example, such forms as liquid formulations, tablets, capsules, and gelcaps. Preferably the dosage forms are solid dosage forms, particularly, tablets comprising about 1 to about 20 mg of a selective PDE5 inhibitor. Any pharmaceutically acceptable excipients for oral use are suitable for preparation of such dosage forms. Suitable pharmaceutical dosage forms include coprecipitate forms described, for example, in Butler U.S. Pat. No. 5,985,326, incorporated herein by reference. In preferred embodiments, the unit dosage form of the present invention is a solid free of a coprecipitate form of the PDE5 inhibitor, but rather contains a solid PDE5 inhibitor as a free drug.
Preferably, the tablets comprise pharmaceutical excipients generally recognized as safe such as lactose, microcrystalline cellulose, starch, calcium carbonate, magnesium stearate, stearic acid, talc, and colloidal silicon dioxide, and are prepared by standard pharmaceutical manufacturing techniques as described in Remington""s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Such techniques include, for example, wet granulation followed by drying, milling, and compression into tablets with or without film coating; dry granulation followed by milling, compression into tablets with or without film coating; dry blending followed by compression into tablets, with or without film coating; molded tablets; wet granulation, dried and filled into gelatin capsules; dry blend filled into gelatin capsules; or suspension and solution filled into gelatin capsules. Generally, the solid dosage forms have identifying marks which are debossed or imprinted on the surface.
The present invention is based on detailed experiments and clinical trials, and the unexpected observations that side effects previously believed to be indicative of PDE5 inhibition can be reduced to clinically insignificant levels by the selection of a selective PDE5 inhibitor having the specific characteristics outlined herein, namely:
1) at least a 100 fold differential in the IC50 values for the inhibition of PDE5 versus PDE6;
2) at least a 1000 fold differential in the IC50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC50 value for the inhibition of PDE5 less than 10 nM.
This unexpected observation enabled the development of articles of manufacture that incorporate a selective PDE5 inhibitor in about 1 to about 20 mg unit dosage forms that, when orally administered, minimize undesired side effects previously believed unavoidable. These side effects include facial flushing, vision abnormalities, and a significant decrease in blood pressure when the PDE5 inhibitor is administered alone or in combination with an organic nitrate. The minimal effect of a present PDE5 inhibitor, administered in about 1 to about 20 mg unit dosage forms, on PDE6 also allows the administration of a selective PDE5 inhibitor to patients suffering from a retinal disease, like diabetic retinopathy or retinitis pigmentosa.
One such selective PDE5 inhibitor, i.e., (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino[1xe2x80x2,2xe2x80x2:1,6]pyrido[3,4-b]indole-1,4-dione, alternatively named (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylene-dioxyphenyl)pyrazino[2xe2x80x2,1xe2x80x2:6,1]pyrido[3,4-b]indole-1,4-dione, is disclosed in Daugan U.S. Pat. No. 5,859,006, and represented by structural formula (I): 
The compound of formula (I) was demonstrated in human clinical studies to exert a minimal impact on systolic blood pressure when administered in conjunction with organic nitrates. By contrast, sildenafil, when administered with nitrates, demonstrates as much as a four-fold greater decrease in systolic blood pressure over a placebo, which leads to the contraindications in the VIAGRA(copyright) insert, and in warnings to certain patients.
Selective PDE5 inhibitors vary significantly in chemical structure, and the use of a selective PDE5 inhibitor as defined in the present invention is not dependent on a particular chemical structure, but rather on the critical parameters outlined herein. However, preferred compounds having the required potency and selectivity can be readily identified by tests described herein from those described in Daugan U.S. Pat. No. 5,859,006, Daugan et al. U.S. Pat. No. 5,981,527, and Daugan et al. U.S. Pat. No. 6,001,847, each of which is incorporated herein by reference.
Preferred compounds of Daugan U.S. Pat. No. 5,859,006 and Daugan et al. U.S. Pat. No. 5,981,527 are represented by structural formula (II): 
wherein R0 is selected from the group consisting of hydrogen, halogen, and C1-6alkyl;
R1 is selected from the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, C1-6alkynyl, halo-C1-6alkyl, C3-8cycloalkyl, C3-8cycloalkylC1-3alkyl, arylC1-3alkyl, wherein aryl is phenyl or phenyl substituted with one to three substituents selected from the group consisting of halogen, C1-6alkyl, C1-6alkoxy, methylenedioxy, and mixtures thereof, and heteroarylC1-3alkyl, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents selected from the group consisting of halogen, C1-6alkyl, C1-6alkoxy, and mixtures thereof;
R2 represents an optionally substituted monocyclic aromatic ring selected from benzene, thiophene, furan, and pyridine, or an optionally substituted bicyclic ring 
attached to the rest of the molecule via one of the benzene ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from the group consisting of oxygen, sulphur and nitrogen;
R3 represents hydrogen or C1-3alkyl, or R1 and R3 together represent a 3- or 4-membered alkyl or alkenyl chain; and salts and solvates thereof.
Other preferred compounds are those of formula (II) wherein:
R0 is hydrogen, halogen, or C1-6alkyl;
R1 is hydrogen or C1-6alkyl;
R2 is the bicyclic ring 
which can be optionally substituted by one or more groups selected from halogen and C1-3alkyl; and
R3 is hydrogen or C1-3alkyl.
The following Table 1 illustrates PDE5 and PDE6 IC50 values for representative selective PDE5 inhibitors disclosed in U.S. Pat. No. 5,859,006, as determined by the procedures described herein.
Compound 5 in Table 1 has the structural formula (I) and additionally demonstrates an IC50 against PDE1c of 10,000 and a ratio of PDE1c/PDE5 of 4,000.
The structures of Compound Nos. 1-5 in Table 1 are as in structural formula (II) wherein R0, R1, R2, and R3 are as follows:
The data in Table 1 indicate that a compound of structural formula (I), wherein R1 is hydrogen or C1-6alkyl, R2 is 
and R3 is hydrogen is especially preferred. Preferably, A is 
Preferred compounds are:
(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)pyrazino[2xe2x80x2,1xe2x80x2:6,1]pyrido[3,4-b]indole-1,4-dione; and
(3S,6R,12aR)-2,3,6,7,12,12a-hexahydro-2,3-dimethyl-6-(3,4-methylenedioxyphenyl)pyrazino[2xe2x80x2,1xe2x80x2:6,1]-pyrido[3,4-b]indole-1,4-dione; and physiologically acceptable salts and solvates (e.g., hydrates) thereof.
Other exemplary compounds useful in the present invention are those disclosed in Daugan et al. U.S. Pat. No. 6,001,847 and WO 97/43287, incorporated herein by reference.
Further exemplary compounds for use in the present invention are disclosed PCT application PCT/EP98/06050, which designates the U.S., entitled xe2x80x9cChemical Compounds,xe2x80x9d inventors A. Bombrun and F. Gellibert, the disclosure of which is specifically incorporated herein by reference. This class of compounds has the structural formula (III): 
and salts and solvates (e.g., hydrates) wherein
C represents a 5- or 6-membered heteroaryl group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
R12 represents hydrogen or halogen;
R13 is selected from the group consisting of
hydrogen,
nitro (NO2),
trifluoromethyl,
trifluoromethoxy,
halogen,
cyano (CN),
a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulphur, optionally substituted with C(xe2x95x90O)ORa or C1-4alkyl,
C1-6alkyl optionally substituted with ORh,
C1-3alkoxy,
C(xe2x95x90O)Rh,
OC(xe2x95x90O)ORh,
C(xe2x95x90O)ORh,
C1-4alkyleneHet,
C1-4alkyleneC(xe2x95x90O)ORh,
OC1-4alkyleneC(xe2x95x90O)ORh,
C1-4alkyleneOC1-4alkyleneC(xe2x95x90O)ORh,
C(xe2x95x90O)NRiSO2Rj,
C(xe2x95x90O)C1-4alkyleneHet,
C1-4alkyleneNRhRi,
C2-6alkenyleneNRhRi,
C(xe2x95x90O)NRhRi,
C(xe2x95x90O)NRhRi,
C(xe2x95x90O)NRhC1-4alkyleneORi,
C(xe2x95x90O)NRhC1-4alkyleneHet,
ORi,
OC2-4alkyleneNRhRi,
OC2-4alkyleneCH(ORh)CH2NRhRi,
OC1-4alkyleneHet,
OC2-4alkyleneORh,
OC2-4alkyleneNRhC(xe2x95x90O)ORh,
NRhRi,
NRhC1-4alkyleneNRhRi,
NRhC(xe2x95x90O)Ri,
NRhC(xe2x95x90O)NRhRi,
N(SO2C1-4alkyl)2,
NRh(SO2C1-4alkyl),
SO2NRhRi,
and OSO2trifluoromethyl;
R14 is selected from the group consisting of hydrogen, halogen, ORh, C1-6alkyl, NO2, and NRhRi;
or R13 and R14 are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
R15 is selected from the group consisting of hydrogen, halogen, NO2, trifluoromethoxy, C1-6alkyl, OC1-6alkyl, and C(xe2x95x90O)ORh;
R16 is hydrogen,
or R15 and R16 are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
Het represents a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with C1-4alkyl;
Rh and Ri can be the same or different and are independently selected from hydrogen and C1-6alkyl;
Rj represents phenyl or C4-6cycloalkyl, wherein the phenyl or C4-6cycloalkyl can be optionally substituted with one or more halogen atoms, one or more C(xe2x95x90O)ORh, or one or more ORh;
n is an integer 1, 2, or 3; and
m is an integer 1 or 2.
Preparations
Human PDE5 Preparation
Recombinant production of human PDE5 was carried out essentially as described in Example 7 of U.S. Pat. No. 5,702,936, incorporated herein by reference, except that the yeast transformation vector employed, which is derived from the basic ADH2 plasmid described in V. Price et al., Methods in Enzymology, 1985, pages 308-318 (1990), incorporated yeast ADH2 promoter and terminator sequences rather than ADH1 promoter and terminator sequences and the Saccharomyces cerevisiase host was the protease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with the American Type Culture Collection, Manassas, Va., under accession number ATCC 74465. Transformed host cells were grown in 2xc3x97SC-leu medium, pH 6.2, with trace metals, and vitamins. After 24 hours, YEP medium containing glycerol was added to a final concentration of 2xc3x97YEP/3% glycerol. Approximately 24 hours later, cells were harvested, washed, and stored at xe2x88x9270xc2x0 C.
Cell pellets (29 g) were thawed on ice with an equal volume of lysis buffer (25 mM Tris-Cl, pH 8, 5 mM MgCl2, 0.25 mM dithiothreitol, 1 mM benzamidine, and 10 xcexcM ZnSO4). Cells were lysed in a microfluidizer with N2 at 20,000 psi. The lysate was centrifuged and filtered through 0.45 xcexcm disposable filters. The filtrate was applied to a 150 mL column of Q Sepharose Fast Flow (Pharmacia). The column was washed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl2, 0.25 mM dithiothreitol, 10 xcexcM ZnSO4) and eluted with a step gradient of 125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl in Buffer A.
Active fractions from the linear gradient were applied to a 180 mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl2, 0.25 mM dithiothreitol, 10 xcexcM ZnSO4, and 250 mM KCl). After loading, the column was washed with 2 volumes of Buffer B and eluted with a linear gradient of 0-125 mM potassium phosphate in Buffer B. Active fractions were pooled, precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mM NaCl, 0.5 mM dithiothreitol, and 10 xcexcM ZnSO4). The pool was applied to a 140 mL column of Sephacryl S-300 HR and eluted with Buffer C. Active fractions were diluted to 50% glycerol and stored at xe2x88x9220xc2x0 C. The resultant preparations were about 85% pure by SDS-PAGE.
Assay for PDE Activity
Activity of PDE5 can be measured by standard assays in the art. For example, specific activity of any PDE can be determined as follows. PDE assays utilizing a charcoal separation technique were performed essentially as described in Loughney et al., (1996), The Journal of Biological Chemistry, 271:796-806. In this assay, PDE5 activity converts [32P]cGMP to [32P]5xe2x80x2GMP in proportion to the amount of PDE5 activity present. The [32P]5xe2x80x2GMP then is quantitatively converted to free [32P] phosphate and unlabeled adenosine by the action of snake venom 5xe2x80x2-nucleotidase. Hence, the amount of [32P] phosphate liberated is proportional to enzyme activity. The assay is performed at 30xc2x0 C. in a 100 xcexcL reaction mixture containing (final concentrations) 40 mM Tris-Cl (pH 8.0), 1 xcexcM ZnSO4, 5 mM MgCl2, and 0.1 mg/mL bovine serium albumin. PDE5 is present in quantities that yield  less than 30% total hydrolysis of substrate (linear assay conditions). The assay is initiated by addition of substrate (1 mM [32P]cGMP), and the mixture is incubated for 12 minutes. Seventy-five (75) xcexcg of Crotalus atrox venom then is added, and the incubation is continued for 3 more minutes (15 minutes total). The reaction is stopped by addition of 200 mL of activated charcoal (25 mg/mL suspension in 0.1 M NaH2PO4, pH 4). After centrifugation (750xc3x97g for 3 minutes) to sediment the charcoal, a sample of the supernatant is taken for radioactivity determination in a scintillation counter and the PDE5 activity is calculated. The preparations had specific activities of about 3 xcexcmoles cGMP hydrolyzed per minute per milligram protein.
Bovine PDE6 Preparation
Bovine PDE6 was supplied by Dr. N. Virmaux, INSERM U338, Strasbourg. Bovine retinas were prepared as described by Virmaux et al., FEBS Letters, 12(6), pp. 325-328 (1971) and see also, A. Sitaramayya et al., Exp. Eye Res., 25, pp. 163-169 (1977). Briefly, unless stated otherwise, all operations were done in the cold and in dim red light. Eyes were kept in the cold and in the dark for up to four hours after slaughtering.
Preparation of bovine retinal outer segment (ROS) basically followed procedures described by Schichi et al., J. Biol. Chem., 224:529 (1969). In a typical experiment, 35 bovine retinas were ground in a mortar with 35 mL 0.066 M phosphate buffer, pH 7.0, made up to 40% with sucrose, followed by homogenization in a Potter homogenizer (20 up and down strokes). The suspension was centrifuged at 25,000xc3x97g for 20 minutes. The pellet was homogenized in 7.5 mL 0.006 M phosphate buffer (40% in sucrose), and carefully layered under 7.5 mL of phosphate buffer (containing no sucrose). Centrifugation was conducted in a swing-out rotor at 45,000xc3x97g for 20 minutes, and produced a pellet which is black at the bottom, and also a red band at the interface 0.066 M. phosphatexe2x80x9440% sucrose/0.066 M phosphate (crude ROS). The red material at the interface was removed, diluted with phosphate buffer, spun down to a pellet, and redistributed in buffered 40% sucrose as described above. This procedure was repeated 2 or 3 times until no pellet was formed. The purified ROS was washed in phosphate buffer and finally spun down to a pellet at 25,000xc3x97g for 20 minutes. All materials were then kept frozen until used.
Hypotonic extracts were prepared by suspending isolated ROS in 10 mM Tris-Cl pH 7.5, 1 mM EDTA, and 1 mM dithioerythritol, followed by centrifugation at 100,000xc3x97g for 30 minutes.
The preparation was reported to have a specific activity of about 35 nmoles cGMP hydrolyzed per minute per milligram protein.
PDE1c Preparation from Spodoptera fugiperda Cells (Sf9)
Cell pellets (5g) were thawed on ice with 20 ml of Lysis Buffer (50 mM MOPS pH 7.4, 10 xcexcM ZnSO4, 0.1 mM CaCl2, 1 mM DTT, 2 mM benzamidine HCl, 5xcexcg/ml each of pepstatin, leupeptin, and aprotenin). Cells were lysed by passage through a French pressure cell (SLM-Aminco) while temperatures were maintained below 10xc2x0 C. The resultant cell homogenate was centrifuged at 36,000 rpm at 4xc2x0 C. for 45 minutes in a Beckman ultracentrifuge using a Type TI45 rotor. The supernatant was discarded and the resultant pellet was resuspended with 40 ml of Solubilization Buffer (Lysis Buffer containing 1M NaCl, 0.1M MgCl2, 1 mM CaCl2, 20 xcexcg/ml calmodulin, and 1% Sulfobetaine SB12 (Z3-12) by sonicating using a VibraCell tuner with a microtip for 3xc3x9730 seconds. This was performed in a crushed ice/salt mix for cooling. Following sonication, the mixture was slowly mixed for 30 minutes at 4xc2x0 C. to finish solubilizing membrane bound proteins. This mixture was centrifuged in a Beckman ultracentrifuge using a type TI45 rotor at 36,000 rpm for 45 minutes. The supernatant was diluted with Lysis Buffer containing 10 xcexcg/ml calpain inhibitor I and II. The precipitated protein was centrifuged for 20 minutes at 9,000 rpm in a Beckman JA-10 rotor. The recovered supernatant then was subjected to Mimetic Blue AP Agarose Chromatography.
In order to run the Mimetic Blue AP Agarose Column, the resin initially was shielded by the application of 10 bed volumes of 1% polyvinyl-pyrrolidine (i.e., MW of 40,000) to block nonspecific binding sites. The loosely bound PVP-40 was removed by washing with 10 bed volumes of 2M NaCl, and 10 mM sodium citrate pH 3.4. Just prior to addition of the solubilized PDE1c sample, the column was equilibrated with 5 bed volumes of Column Buffer A (50 mM MOPS pH 7.4, 10 xcexcM ZnSO4, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM DTT, 2 mM benzamidine HCl).
The solubilized sample was applied to the column at a flow rate of 2 ml/min with recycling such that the total sample was applied 4 to 5 times in 12 hours. After loading was completed, the column was washed with 10 column volumes of Column Buffer A, followed by 5 column volumes of Column Buffer B (Column Buffer A containing 20 mM 5xe2x80x2-AMP), and followed by 5 column volumes of Column Buffer C (50 mM MOPS pH 7.4, 10 xcexcM ZnSO4, 0.1 mM CaCl2, 1 mM dithiothreitol, and 2 mM benzamidine HCl). The enzyme was eluted into three successive pools. The first pool consisted of enzyme from a 5 bed volume wash with Column Buffer C containing 1 mM cAMP. The second pool consisted of enzyme from a 10 bed volume wash with Column Buffer C containing 1 M NaCl. The final pool of enzyme consisted of a 5 bed volume wash with Column Buffer C containing 1 M NaCl and 20 mM cAMP.
The active pools of enzyme were collected and the cyclic nucleotide removed via conventional gel filtration chromatography or chromatography on hydroxy-apatite resins. Following removal of cyclic nucleotides, the enzyme pools were dialyzed against Dialysis Buffer containing 25 mM MOPS pH 7.4, 10 xcexcM ZnSO4, 500 mM NaCl, 1 mM CaCl2, 1 mM dithiothreitol, 1 mM benzamidine HCl, followed by dialysis against Dialysis buffer containing 50% glycerol. The enzyme was quick frozen with the aid of dry ice and stored at xe2x88x9270xc2x0 C.
The resultant preparations were about  greater than 90% pure by SDS-PAGE. These preparations had specific activities of about 0.1 to 1.0 xcexcmol cAMP hydrolyzed per minute per milligram protein.
IC50 Value Determinations
The parameter of interest in evaluating the potency of a competitive enzyme inhibitor of PDE5 and/or PDE1c and PDE6 is the inhibition constant, i.e., Ki. This parameter can be approximated by determining the IC50, which is the inhibitor concentration that results in 50% enzyme inhibition, in a single dose-response experiment under the following conditions.
The concentration of inhibitor is always much greater than the concentration of enzyme, so that free inhibitor concentration (which is unknown) is approximated by total inhibitor concentration (which is known).
A suitable range of inhibitor concentrations is chosen (i.e., inhibitor concentrations at least several fold greater and several fold less than the Kiare present in the experiment). Typically, inhibitor concentrations ranged from 10 nM to 10 xcexcM.
The concentrations of enzyme and substrate are chosen such that less than 20% of the substrate is consumed in the absence of inhibitor (providing, e.g., maximum substrate hydrolysis of from 10 to 15%), so that enzyme activity is approximately constant throughout the assay.
The concentration of substrate is less than one-tenth the Michaelis constant (Km). Under these conditions, the IC50 will closely approximate the Ki. This is because of the Cheng-Prusoff equation relating these two parameters: IC50=Ki (1+S/Km), with (1+S/Km) approximately 1 at low values of S/Km.
The IC50 value is estimated from the data points by fitting the data to a suitable model of the enzyme inhibitor interaction. When this interaction is known to involve simple competition of the inhibitor with the substrate, a two-parameter model can be used:
Y=A/(1+x/B)
where the y is the enzyme activity measured at an inhibitor concentration of x, A is the activity in the absence of inhibitor and B is the IC50. See Y. Cheng et al., Biochem. Pharmacol., 22:3099-3108 (1973).
Effects of inhibitors of the present invention on enzymatic activity of PDE5 and PDE6 preparations as described above were assessed in either of two assays which differed from each other principally on the basis of scale and provided essentially the same results in terms of IC50 values. Both assays involved modification of the procedure of Wells et al., Biochim. Biophys. Acta, 384:430 (1975). The first of the assays was performed in a total volume of 200 xcexcl containing 50 mM Tris pH 7.5, 3 mM Mg acetate, 1 mM EDTA, 50 xcexcg/mL snake venom nucleotidase and 50 nM [3H]-cGMP (Amersham). Compounds of the invention were dissolved in DMSO finally present at 2% in the assay. The assays were incubated for 30 minutes at 30xc2x0 C. and stopped by addition of 800 xcexcl of 10 mM Tris pH 7.5, 10 mM EDTA, 10 mM theophylline, 0.1 mM adenosine, and 0.1 mM guanosine. The mixtures were loaded on to 0.5 mL QAE Sephadex columns, and eluted with 2 mL of 0.1 M formate (pH 7.4). The eluted radioactivity was measured by scintillation counting in Optiphase Hisafe 3.
A second, microplate, PDE assay was developed using Multiscreen plates and a vacuum manifold. The assay (100 xcexcl) contained 50 mM Tris pH 7.5, 5 mM Mg acetate, 1 mM EDTA and 250 xcexcg/mL snake venom nucleotidase. The other components of the reaction mixture were as described above. At the end of the incubation, the total volume of the assays were loaded on a QAE Sephadex microcolumn plate by filtration. Free radioactivity was eluted with 200 xcexcl of water from which 50 xcexcl aliquots were analyzed by scintillation counting as described above.
The following examples are presented to further illustrate the preparation of the claimed invention. The scope of the present invention is not to be construed as merely consisting of the following examples.